Gene Editing

  1. Gene Editing Workflow and Implementation
    1. Experimental Design and Planning
      1. Target Site Selection
        1. Functional Considerations
          1. Accessibility Assessment
            1. PAM Availability
              1. Conservation Analysis
              2. Guide RNA Design
                1. Computational Design Tools
                  1. Specificity Prediction
                    1. Efficiency Scoring
                      1. Off-target Analysis
                      2. Experimental Controls
                        1. Negative Controls
                          1. Positive Controls
                            1. Mock Treatments
                            2. Donor Template Design
                              1. Homology Arm Design
                                1. Insert Sequence Optimization
                                  1. Template Format Selection
                                2. Delivery Methods and Strategies
                                  1. Viral Delivery Systems
                                    1. Adeno-Associated Viruses
                                      1. Serotype Selection
                                        1. Packaging Capacity Limitations
                                          1. Tissue Tropism
                                            1. Safety Profile
                                            2. Lentiviruses
                                              1. Integration Characteristics
                                                1. Packaging Systems
                                                  1. Safety Considerations
                                                  2. Adenoviruses
                                                    1. High Transduction Efficiency
                                                      1. Immunogenicity Concerns
                                                    2. Non-Viral Delivery Methods
                                                      1. Electroporation
                                                        1. Mechanism and Protocols
                                                          1. Cell Type Optimization
                                                            1. Nucleofection Technology
                                                            2. Lipid-based Delivery
                                                              1. Lipofection Reagents
                                                                1. Lipid Nanoparticles
                                                                  1. Formulation Optimization
                                                                  2. Physical Delivery Methods
                                                                    1. Microinjection
                                                                      1. Gene Gun
                                                                        1. Sonoporation
                                                                        2. Chemical Delivery
                                                                          1. Calcium Phosphate
                                                                            1. Polyethylenimine
                                                                          2. In Vivo Delivery Considerations
                                                                            1. Target Tissue Accessibility
                                                                              1. Systemic vs. Local Delivery
                                                                                1. Immune Response Management
                                                                                  1. Delivery Efficiency Optimization
                                                                                2. Experimental Validation and Analysis
                                                                                  1. On-Target Editing Assessment
                                                                                    1. PCR-based Analysis
                                                                                      1. Primer Design
                                                                                        1. Amplicon Sequencing
                                                                                        2. Restriction Enzyme Analysis
                                                                                          1. RFLP Assays
                                                                                            1. T7 Endonuclease I Assays
                                                                                            2. Sequencing Methods
                                                                                              1. Sanger Sequencing
                                                                                                1. Next-Generation Sequencing
                                                                                                  1. Long-read Sequencing
                                                                                                2. Off-Target Analysis
                                                                                                  1. Computational Prediction
                                                                                                    1. Algorithm Development
                                                                                                      1. Genome-wide Scanning
                                                                                                      2. Experimental Detection
                                                                                                        1. GUIDE-seq
                                                                                                          1. CIRCLE-seq
                                                                                                            1. DISCOVER-seq
                                                                                                              1. Digenome-seq
                                                                                                              2. Validation of Predicted Sites
                                                                                                              3. Functional Validation
                                                                                                                1. Phenotypic Analysis
                                                                                                                  1. Morphological Changes
                                                                                                                    1. Behavioral Assessments
                                                                                                                    2. Molecular Analysis
                                                                                                                      1. Protein Expression
                                                                                                                        1. RNA Expression
                                                                                                                          1. Pathway Analysis
                                                                                                                          2. Cellular Function Tests
                                                                                                                            1. Proliferation Assays
                                                                                                                              1. Differentiation Studies

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                                                                                                                        6. Variations and Innovations in CRISPR Technology

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                                                                                                                        8. Applications in Biomedical Research